Immunization of mice against Naegleria fowleri infection.

Naegleria fowleri produces fatal meningoencephalitis in humans and in experimentally infected laboratory animals. The course of the disease in mice is dependent upon the infecting dose of amoebae, route of inoculation, and prior exposure to Naegleria antigens. DUB/ICR mice were immunized by various routes and antigen preparations, held for 21 days, and, together with noninfected control mice, challenged intravenously (i.v.) or intranasally (i.n.) with 10(7) or 10(6) N. fowleri per mouse, respectively. Mice immunized with liver or formalinized N. fowleri or live N. gruberi subcutaneously, intraperitoneally, i.v., or i.n. were significantly protected against a subsequent lethal challenge with N. fowleri i.v. or i.n. In general, i.v. inoculation afforded greated protection than other routes of immunization, intact cells immunized mice better than did cell fragments, and N. gruberi appeared to be a better immunogen than N. fowleri.     

Innate resistance of mice to experimental infection with Naegleria fowleri.

The mouse system provides an excellent model for studying host resistance to Naegleria fowleri, the agent of primary amoebic meningoencephalitis. Innate resistance to infection with N. fowleri was examined with respect to infecting dose and the age, sex, and strain of mice. Intravenous inoculation with 10(7) amoebae per mouse produced 100% mortality in 9 days, whereas inoculation with fewer amoebae reduced the cumulative mortality. Male and female DUB/ICR mice of varying ages were inoculated intravenously with 2.5 X 10(5) N. fowleri per g of body weight. The youngest mice died first, with 100% mortality for both males and females, and mortality decreased with increasing age. Female mice were significantly more resistant to infection than males. Five strains of mice weighing approximately 20 g were inoculated intravenously with weight-adjusted doses; mortality ranged from 10% in C57BL/6 mice to 95% in A/HeCr mice.     

Role of the Nfa1 protein in pathogenic Naegleria fowleri cocultured with CHO target cells

Naegleria fowleri, a free-living amoeba, exists as a virulent pathogen which causes fatal primary amoebic meningoencephalitis in experimental animals and humans. Using infected and immune mouse sera, we previously cloned an nfa1 gene from a cDNA library of N. fowleri by immunoscreening. The nfa1 gene (360 bp) produced a recombinant 13.1-kDa protein, and the Nfa1 protein showed pseudopodium-specific immunolocalization on a trophozoite of N. fowleri. In this study, the role of the Nfa1 protein as a cell contact mechanism of N. fowleri cocultured with target cells was observed by an immunofluorescence assay with an anti-Nfa1 polyclonal antibody. Using confocal microscopic findings, the Nfa1 protein was located on the pseudopodia of N. fowleri trophozoites. The Nfa1 protein in N. fowleri trophozoites cocultured with CHO target cells was also located on pseudopodia, as well as in a food cup formed as a phagocytic structure in close contact with target cells. The amount of nfa1 mRNA of N. fowleri was strongly increased 6 h after coculture.     

Depression of immunity to Naegleria fowleri in mice by selective depletion of neutrophils with a monoclonal antibody.

In an attempt to define the role of neutrophils in immunity to Naegleria fowleri in vivo, we examined the effects of treating immunized (with amoeba culture supernatant antigen) mice with the monoclonal antibody NIMP-R10, which binds to neutrophil complement receptor type 3bi (CR3) and causes selective neutrophil depletion in mice. Mice in the nonimmunized group challenged with amoebae all died by day 12, while 97% in the immunized group survived. By contrast, the immunized group treated with NIMP-R10 showed only 25% survival. The immunized group treated with "control" mouse ascites, WEM-G11, was highly resistant (90% survival). There was a significant neutrophil response in the nasal mucosa and olfactory lobes of immunized, NIMP-R10-treated mice, despite a marked degree of neutropenia similar to that seen in immunized, untreated mice. Nonimmunized mice showed virtually no neutrophil response. Despite this response in the NIMP-R10-treated mice, amoebic proliferation was not depressed, and there was no evidence of neutrophil degranulation or amoebic killing, despite the close apposition of large numbers of neutrophils to amoebae. The results indicate that neutrophils are necessary for the expression of immunity to N. fowleri.     

Intranasal coadministration of the Cry1Ac protoxin with amoebal lysates increases protection against Naegleria fowleri meningoencephalitis

Cry1Ac protoxin has potent mucosal and systemic adjuvant effects on antibody responses to proteins or polysaccharides. In this work, we examined whether Cry1Ac increased protective immunity against fatal Naegleria fowleri infection in mice, which resembles human primary amoebic meningoencephalitis. Higher immunoglobulin G (IgG) than IgA anti-N. fowleri responses were elicited in the serum and tracheopulmonary fluids of mice immunized by the intranasal or intraperitoneal route with N. fowleri lysates either alone or with Cry1Ac or cholera toxin. Superior protection against a lethal challenge with 5 x 10(4) live N. fowleri trophozoites was achieved for immunization by the intranasal route. Intranasal immunization of N. fowleri lysates coadministered with Cry1Ac increased survival to 100%; interestingly, immunization with Cry1Ac alone conferred similar protection to that achieved with amoebal lysates alone (60%). When mice intranasally immunized with Cry1Ac plus lysates were challenged with amoebae, both IgG and IgA mucosal responses were rapidly increased, but only the increased IgG response persisted until day 60 in surviving mice. The brief rise in the level of specific mucosal IgA does not exclude the role that this isotype may play in the early defense against this parasite, since higher IgA responses were detected in nasal fluids of mice intranasally immunized with lysates plus either Cry1Ac or cholera toxin, which, indeed, were the treatments that provided the major protection levels. In contrast, serum antibody responses do not seem to be related to the protection level achieved. Both acquired and innate immune systems seem to play a role in host defense against N. fowleri infection, but further studies are required to elucidate the mechanisms involved in protective effects conferred by Cry1Ac, which may be a valuable tool to improve mucosal vaccines.     

The immune response induced by DNA vaccine expressing nfa1 gene against Naegleria fowleri

The pathogenic free-living amoeba, Naegleria fowleri, causes fatal primary amoebic meningoencephalitis in experimental animals and in humans. The nfa1 gene that was cloned from N. fowleri is located on pseudopodia, especially amoebic food cups and plays an important role in the pathogenesis of N. fowleri. In this study, we constructed and characterized retroviral vector and lentiviral vector systems for nfa1 DNA vaccination in mice. We constructed the retroviral vector (pQCXIN) and the lentiviral vector (pCDH) cloned with the egfp-nfa1 gene. The expression of nfa1 gene in Chinese hamster ovary cell and human primary nasal epithelial cell transfected with the pQCXIN/egfp-nfa1 vector or pCDH/egfp-nfa1 vector was observed by fluorescent microscopy and Western blotting analysis. Our viral vector systems effectively delivered the nfa1 gene to the target cells and expressed the Nfa1 protein within the target cells. To evaluate immune responses of nfa1-vaccinated mice, BALB/c mice were intranasally vaccinated with viral particles of each retro- or lentiviral vector expressing nfa1 gene. DNA vaccination using viral vectors expressing nfa1 significantly stimulated the production of Nfa1-specific IgG subclass, as well as IgG levels. In particular, both levels of IgG2a (Th1) and IgG1 (Th2) were significantly increased in mice vaccinated with viral vectors. These results show the nfa1-vaccination induce efficiently Th1 type, as well as Th2 type immune responses. This is the first report to construct viral vector systems and to evaluate immune responses as DNA vaccination in N. fowleri infection. Furthermore, these results suggest that nfal vaccination may be an effective method for treatment of N. fowleri infection.     

Vaccination with Lentiviral Vector Expressing the nfa1 Gene Confers a Protective Immune Response to Mice Infected with Naegleria fowleri

Naegleria fowleri, a pathogenic free-living amoeba, causes fatal primary amoebic meningoencephalitis (PAM) in humans and animals. The nfa1 gene (360 bp), cloned from a cDNA library of N. fowleri, produces a 13.1-kDa recombinant protein which is located on pseudopodia, particularly the food cup structure. The nfa1 gene plays an important role in the pathogenesis of N. fowleri infection. To examine the effect of nfa1 DNA vaccination against N. fowleri infection, we constructed a lentiviral vector (pCDH) expressing the nfa1 gene. For the in vivo mouse study, BALB/c mice were intranasally vaccinated with viral particles of a viral vector expressing the nfa1 gene. To evaluate the effect of vaccination and immune responses of mice, we analyzed the IgG levels (IgG, IgG1, and IgG2a), cytokine induction (interleukin-4 [IL-4] and gamma interferon [IFN-γ]), and survival rates of mice that developed PAM. The levels of both IgG and IgG subclasses (IgG1 and IgG2a) in vaccinated mice were significantly increased. The cytokine analysis showed that vaccinated mice exhibited greater IL-4 and IFN-γ production than the other control groups, suggesting a Th1/Th2 mixed-type immune response. In vaccinated mice, high levels of Nfa1-specific IgG antibodies continued until 12 weeks postvaccination. The mice vaccinated with viral vector expressing the nfa1 gene also exhibited significantly higher survival rates (90%) after challenge with N. fowleri trophozoites. Finally, the nfa1 vaccination effectively induced protective immunity by humoral and cellular immune responses in N. fowleri-infected mice. These results suggest that DNA vaccination using a viral vector may be a potential tool against N. fowleri infection.     

Protective immunity to systemic nocardiosis in mice immunized with cell extract antigens ofNocardia asteroides

Mice immunized with crude cell extract antigens ofNocardia asteroides as well as its purified fraction F₁ elicited significant levels of both humoral and cellular immune responses. These animals when challenged with 50% lethal dose ofN. asteroides, two weeks after the complete immunization exhibited significant protection as seen by decreased mortality and viable counts compared to nonimmunized. The immunized animals were capable of clearing the infection much faster as compared to control. Cell extract antigens which were mainly protein in nature are thought to have a prime role in prevention and restriction of nocardial infection.     

Identification of a Naegleria fowleri membrane protein reactive with anti-human CD59 antibody

Naegleria fowleri, the causative agent of primary amebic meningoencephalitis, is resistant to complement lysis. The presence of a complement regulatory protein on the surface of N. fowleri was investigated. Southern blot and Northern blot analyses demonstrated hybridization of a radiolabeled cDNA probe for CD59 to genomic DNA and RNA, respectively, from pathogenic N. fowleri. An 18-kDa immunoreactive protein was detected on the membrane of N. fowleri by Western immunoblot and immunofluorescence analyses with monoclonal antibodies for human CD59. Complement component C9 immunoprecipitated with the N. fowleri "CD59-like" protein from amebae incubated with normal human serum. In contrast, a gene or protein similar to CD59 was not detected in nonpathogenic, complement-sensitive N. gruberi amebae. Collectively, our studies suggest that a protein reactive with antibodies to human CD59 is present on the surface of N. fowleri amebae and may play a role in resistance to lysis by cytolytic proteins.     

Protective immunity induced by Toxoplasma gondii rhoptry protein 16 against toxoplasmosis in mice

Toxoplasma gondii can infect a large variety of domestic and wild animals and human beings, sometimes causing severe pathology. Rhoptries are involved in T. gondii invasion and host cell interaction and have been implicated as important virulence factors. In this study, we constructed a DNA vaccine expressing rhoptry protein 16 (ROP16) of T. gondii and evaluated the immune responses it induced in Kunming mice. The gene sequence encoding ROP16 was inserted into the eukaryotic expression vector pVAX I. We immunized Kunming mice intramuscularly. After immunization, we evaluated the immune response using a lymphoproliferative assay, cytokine and antibody measurements, and the survival times of mice challenged lethally. The results showed that mice immunized with pVAX-ROP16 developed a high level of specific antibody responses against T. gondii ROP16 expressed in Escherichia coli, a strong lymphoproliferative response, and significant levels of gamma interferon (IFN-γ), interleukin-2 (IL-2), IL-4, and IL-10 production compared with results for other mice immunized with either empty plasmid or phosphate-buffered saline, respectively. The results showed that pVAX-ROP16 induces significant humoral and cellular Th1 immune responses. After lethal challenge, the mice immunized with pVAX-ROP16 showed a significantly (P < 0.05) prolonged survival time (21.6 ± 9.9 days) compared with control mice, which died within 7 days of challenge. Our data demonstrate, for the first time, that ROP16 triggers a strong humoral and cellular response against T. gondii and that ROP16 is a promising vaccine candidate against toxoplasmosis, worth further development.     

Activation of a heat-stable cytolytic protein associated with the surface membrane of Naegleria fowleri.

Surface membrane-enriched fractions of Naegleria fowleri obtained after isopycnic centrifugation experiments contain a potent cytolytic activity as determined by hemolysis and 51Cr release assays. This surface membrane cytolysin was unaffected by a treatment at 75 degrees C for 30 min and accounted for 70 to 90% of cytolysis by whole-cell lysates of amoebae. This heat resistance as well as intimate membrane association distinguished the surface membrane cytolytic activity from a second heat-labile cytolytic activity which appears to be latent within lysosomes. The surface membrane cytolysin was found to be specifically activated by diluted samples of lysosomal fractions. The possible role of this surface membrane cytotoxin in the pathogenicity of N. fowleri is discussed.     

耐甲氧西林金黄色葡萄球菌(MRSA)重组亚单位疫苗FnBPA的免疫保护作用研究

目的评价耐甲氧西林金黄色葡萄球菌(MRSA)候选疫苗抗原FnBPA活性片段的免疫原性以及其在抵抗耐甲氧西林金黄色葡萄球菌感染中的免疫保护作用。方法构建pGEX-6p-2-FnBPA表达载体,经可溶表达及亲和纯化后的重组蛋白免疫Balb/c小鼠。末次免疫后的第7天,分离免疫小鼠的血清ELISA检测IgG及其亚型抗体水平,同时测定体外抗体介导的调理吞噬作用。末次免疫后的第14天,经尾静脉感染ATCC国际标准株MRSA-252,统计分析感染后各组的小鼠的生存率以及检测脾脏,肾脏的细菌定植量。结果成功构建、表达及纯化了FnBPA活性片段,重组蛋白纯度可达90%;免疫后诱导小鼠机体产生了高效价的IgG抗体,并在体外能发挥抗体介导的调理吞噬作用;在感染保护评价实验中,与对照组相比,实验组的生存率显著提高(P<0.01),并显著降低了脾脏与肾脏的细菌定植量(P<0.05)。结论耐甲氧西林金黄色葡萄球菌重组亚单位疫苗FnBPA具有良好的免疫原性和免疫保护作用,可以作为MRSA疫苗研究的重要候选抗原。     

Complete protection against lethal Toxoplasma gondii infection in mice immunized with a plasmid encoding the SAG1 gene

Infection with the protozoan parasite Toxoplasma gondii is transmitted to humans from infected animals by tissue cysts and oocysts excreted by cats. Immunization with inactivated parasites or recombinant proteins has at best shown partial protection. We constructed a plasmid expressing the SAG1 surface antigen of T. gondii, p1tPASAG1, and showed that animals immunized with the plasmid produce anti-SAG1 antibodies which recognize the native SAG1. Mice immunized with p1tPASAG1 showed 80 to 100% protection against challenge with the non-cyst-producing, virulent RH isolate, compared to an 80% mortality in mice immunized with empty plasmid, which is the greatest efficacy of any vaccine against T. gondii produced so far. The SAG1 molecule was analyzed for potential cytotoxic T-lymphocyte (CTL) epitopes, and four peptides with the best fit were synthesized. The ability of the peptides to stimulate gamma interferon production by CD8(+) T cells from p1tPASAG1-immunized mice was tested in an ELISPOT assay, and one new CTL epitope was identified. Adoptive transfer of CD8(+) T cells from p1tPASAG1-immunized to na?ve mice showed partial protection. In conclusion, DNA vaccination with p1tPASAG1 gave effective protection in mice against T. gondii infection and the protection could be adoptively transferred by purified CD8(+) T cells.     

Protective immunity against congenital toxoplasmosis with recombinant SAG1 protein in a guinea pig model

Primary infection with Toxoplasma gondii during pregnancy can induce fetal pathology and abortion in both humans and animals. The present study describes the development of an experimental model of congenital toxoplasmosis in the guinea pig. In this animal model, we evaluated the protective effect of vaccination with a recombinant form of SAG1 against maternofetal transmission of tachyzoites. The presence of parasites in fetuses was determined by nested PCRs and by an in vivo readout after fetal brain homogenate injections in mice. The absence of parasites was demonstrated in 66 to 86% of fetuses derived from adult guinea pigs immunized with SAG1 and challenged with the mildly virulent T. gondii strain C56. In contrast, more than 80% of fetuses from mock-immunized guinea pigs were infected. The protection was not correlated with titers of antibody to SAG1. Our results indicated that this experimental model constitutes a relevant model for evaluation of vaccine candidates against congenital toxoplasmosis and that SAG1 elicits significant protection against maternofetal transmission.     

Role of the Nfa1 Protein in Pathogenic Naegleria fowleri Cocultured with CHO Target Cells

Naegleria fowleri, a free-living amoeba, exists as a virulent pathogen which causes fatal primary amoebic meningoencephalitis in experimental animals and humans. Using infected and immune mouse sera, we previously cloned an nfa1 gene from a cDNA library of N. fowleri by immunoscreening. The nfa1 gene (360 bp) produced a recombinant 13.1-kDa protein, and the Nfa1 protein showed pseudopodium-specific immunolocalization on a trophozoite of N. fowleri. In this study, the role of the Nfa1 protein as a cell contact mechanism of N. fowleri cocultured with target cells was observed by an immunofluorescence assay with an anti-Nfa1 polyclonal antibody. Using confocal microscopic findings, the Nfa1 protein was located on the pseudopodia of N. fowleri trophozoites. The Nfa1 protein in N. fowleri trophozoites cocultured with CHO target cells was also located on pseudopodia, as well as in a food cup formed as a phagocytic structure in close contact with target cells. The amount of nfa1 mRNA of N. fowleri was strongly increased 6 h after coculture.     

Mechanisms of defense formation against meningococcal infection in mice immunized with synthetic peptides

Immunization of mice with synthetic peptide fragments of conservative sites of meningococcal outer membrane proteins led to defense formation against infection with virulent serogroup A and B Meningococci. The role of cellular immunity in the formation of defense against meningococcal infection after immunization with the peptides and the possibility of stimulating lymphocyte population with these peptides were demonstrated. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 143, No. 6, pp. 663–666, June, 2007     

New generation of mucosal adjuvants for the induction of protective immunity

Invasion of infectious agents through mucosal surfaces can be prevented by use of the common mucosal immune system (CMIS), which interconnects inductive tissues, including Peyer's patches (PPs) and nasopharyngeal-associated lymphoreticular tissue (NALT), and effector tissues of the intestinal and respiratory tracts. In order for the CMIS to induce maximal protective mucosal immunity, co-administration of mucosal adjuvant has been shown to be essential. When vaccine antigen is administered together with mucosal adjuvant, antigen-specific T-helper (Th) 1 and Th2 cells, cytotoxic T lymphocytes (CTLs) and IgA B cell responses are effectively induced by oral or nasal routes via the CMIS. In the early stages of induction of mucosal immune response, the uptake of orally or nasally administered antigens is achieved through a unique set of antigen-sampling cells, M cells located in follicle-associated epithelium (FAE) of inductive sites. After successful uptake, the antigens are immediately processed and presented by the underlying dendritic cells (DCs). Elucidation of the molecular/cellular characteristics of M cells and mucosal DCs will greatly facilitate the design of a new generation of effective mucosal adjuvants and of a vaccine delivery vehicle that maximises the use of the CMIS. Our recent efforts at mucosal vaccine development have focused on nasal administration of vaccine antigen together with nontoxic mutant-based or cytokine-/chemokine-based adjuvant for the induction of the protective immunity. To this end, a chimeric form of a nontoxic adjuvant combining the merits of mutant cholera toxin A subunit (mCT-A) and heat labile toxin B subunit (LT-B) was created as the second generation of detoxified toxin-based mucosal adjuvant. When a vaccine antigen was coexpressed together with an immune stimulatory/delivery molecule in crop seed, this edible vaccine is not only effective but also extremely practical in that it can be produced in huge quantities and preserved and shipped over long distances at room temperature without altering the quality of the vaccine. Because such qualities would greatly facilitate global vaccination, this new generation edible vaccines with a built-in adjuvant and/or M cell-targeted edible vaccine promises to be a powerful weapon for combating infectious diseases and bioterrorism.     

Protective immunity against Streptococcus mutans infection in mice after intranasal immunization with the glucan-binding region of S. mutans glucosyltransferase

Here we present the construction and characterization of a chimeric vaccine protein combining the glucan-binding domain (GLU) of the gtfB-encoded water-insoluble glucan-synthesizing glucosyltransferase enzyme (GTF-I) from Streptococcus mutans and thioredoxin from Escherichia coli, which increases the solubility of coexpressed recombinant proteins and stimulates proliferation of murine T cells. The protective potential of intranasal (i.n.) immunization with this chimeric immunogen was compared to that of the GLU polypeptide alone in a mouse infection model. Both immunogens were able to induce statistically significant mucosal (salivary and vaginal) and serum responses (P < 0.01) which were sustained to the end of the study (experimental day 100). Following infection with S. mutans, sham-immunized mice maintained high levels of this cariogenic organism ( approximately 60% of the total oral streptococci) for at least 5 weeks. In contrast, animals immunized with the thioredoxin-GLU chimeric protein (Thio-GLU) showed significant reduction (>85%) in S. mutans colonization after 3 weeks (P < 0.05). The animals immunized with GLU alone required 5 weeks to demonstrate significant reduction (>50%) of S. mutans infection (P < 0.05). Evaluation of dental caries activity at the end of the study showed that mice immunized with either Thio-GLU or GLU had significantly fewer carious lesions in the buccal enamel or dentinal surfaces than the sham-immunized animals (P < 0.01). The protective effects against S. mutans colonization and caries activity following i.n. immunization with GLU or Thio-GLU are attributed to the induced salivary immunoglobulin A (IgA) anti-GLU responses. Although in general Thio-GLU was not significantly better than GLU alone in stimulating salivary IgA responses and in protection against dental caries, the finding that the GLU polypeptide alone, in the absence of any immunoenhancing agents, is protective against disease offers a promising and safe strategy for the development of a vaccine against caries.     

The nontoxic CTA1-DD adjuvant enhances protective immunity against Helicobacter pylori infection following mucosal immunization

Safe and efficacious adjuvants are much needed to facilitate the development of mucosal vaccines. Here, we have asked whether our nontoxic vaccine adjuvant, CTA1-DD, can enhance protective immunity against Helicobacter pylori infection. Intranasal immunizations with H. pylori lysate together with CTA1-DD-adjuvant induced significant protection in C57Bl/6 mice, almost as strong as similar immunizations using cholera toxin (CT)-adjuvant. Protection remained strong even at 8 weeks postchallenge and the bacterial colonization was reduced by 20-fold compared to lysate-immunized controls. Although CTA1-DD was designed to bind to B cells, microMT mice developed significant, but lower, level of protection following immunization. Intranasal immunizations with CT adjuvant in C57Bl/6 mice resulted in the development of severe postimmunization gastritis at 2 and 8 weeks postchallenge, whereas the degree of gastritis was substantially lower in the CTA1-DD-immunized mice. Protection induced by both CTA1-DD- and CT adjuvant was associated with a strong local infiltration of CD4(+) T cells in the gastric mucosa, and recall responses to specific Ag elicited substantial IFN-gamma production, indicating Th1-dominance. These findings clearly demonstrate that CTA1-DD adjuvant is a promising candidate to be further exploited in the development of a mucosal vaccine against H. pylori infection.